Method For Simulating The Functional Attributes Of Human Milk Oligosaccharides In Formula-Fed Infants

ABSTRACT

The present invention is directed to a novel method for increasing the production of acetate, decreasing the production of butyrate, increasing the population and species of beneficial bacteria and slowing the rate of fermentation of prebiotics within the gut of a formula-fed infant. The method comprises administration of a therapeutically effective amount of PDX to the infant.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a method for simulating the functionalattributes of human milk oligosaccharides in infants.

(2) Description of the Related Art

The infant gut microflora is rapidly established in the first few weeksfollowing birth. The nature of this intestinal colonization is initiallydetermined by early exposure to environmental sources of microbes aswell as the health of the infant. Whether the infant is breast-fed orformula fed also has a strong influence on the intestinal bacterialpopulation.

In the breast-fed infant, for example, Bifidobacterium spp. dominateamong intestinal bacteria, with Streptococcus spp. and Lactobacillusspp. as less common contributors. In contrast, the microflora offormula-fed infants is more diverse, containing Bifidobacterium spp. andBacteroides spp. as well as the more pathogenic species, Staphylococcus,Escherichia coli and Clostridia. The varied species of Bifidobacteriumin the stools of breast-fed and formula-fed infants differ as well.

Bifidobacteria are generally considered “beneficial” bacteria and areknown to protect against colonization by pathogenic bacteria. Thislikely occurs through competition for cell surface receptors,competition for essential nutrients, production of anti-microbialagents, and production of inhibitory compounds such as short chain fattyacids (SCFA) which may decrease fecal pH and inhibit potentiallypathogenic bacteria. Bifidobacteria are also associated with resistanceto gastrointestinal (GI) tract and respiratory infection as well as anenhanced immune function in children and infants. Therefore, thepromotion of an intestinal environment in which Bifidobacteria dominatehas become a goal in the development of nutritional formulations forformula-fed infants.

Human milk (HM) contains a number of factors that may contribute to thegrowth and population of Bifidobacteria in the gut microflora ofinfants. Among these factors is a complex mixture of more than 130different oligosaccharides that reach levels as high as 8-12 g/L intransitional and mature milk. Kunz, et al., Oligosaccharides in HumanMilk: Structure, Functional, and Metabolic Aspects, Ann. Rev. Nutr. 20:699-722 (2000). These oligosaccharides are resistant to enzymaticdigestion in the upper gastrointestinal tract and reach the colonintact, where they serve as substrates for colonic fermentation.

HM oligosaccharides are believed to elicit an increase in the number ofBifidobacteria in the colonic flora, along with a reduction in thenumber of potentially pathogenic bacteria. Kunz, et al.,Oligosaccharides in Human Milk: Structure, Functional, and MetabolicAspects, Ann. Rev. Nutr. 20: 699-722 (2000); Newburg, Do the BindingProperties of Oligosaccharides in Milk Protect Human Infants fromGastrointestinal Bacteria?, J. Nutr. 217:S980-S984 (1997). One way thatHM oligosaccharides may increase the number of Bifidobacteria and reducethe number of potentially pathogenic bacteria is by acting ascompetitive receptors and inhibiting the binding of pathogens to thecell surface. Rivero-Urgell, et al., Oligosaccharides: Application inInfant Food, Early Hum. Dev. 65(S):43-52 (2001).

In addition to reducing the number of pathogenic bacteria and promotingthe population of bifidobacteria, when HM oligosaccharides arefermented, they produce SCFAs such as acetic, propionic and butyricacids. These SCFAs are believed to contribute to caloric content, serveas a major energy source for the intestinal epithelium, stimulate sodiumand water absorption in the colon, and enhance small bowel digestion andabsorption. In addition, SCFA are believed to contribute to overallgastrointestinal health by modulating gastrointestinal development andimmune function.

The fermentation of HM oligosaccharides also reduces fecal ammonia,amine, and phenol concentrations, which have been implicated as themajor odorous components of feces. Cummings & Macfarlane, The Controland Consequences of Bacterial Fermentation in the Human Colon, J. Appl.Bacteriol. 70:443-459 (1991); Miner & Hazen, Ammonia and Amines:Components of Swine-Building Odor ASAE 12:772-774 (1969); Spoelstra,Origin of Objectionable Components in Piggery Wastes and the Possibilityof Applying Indicator Components for Studying Odour Development, Agric.Environ. 5:241-260 (1980); O'Neill & Phillips, A Review of the Controlof Odor Nuisance from Livestock Buildings: Part 3. Properties of theOdorous Substances which have been Identified in Livestock Wastes or inthe Air Around them J. Agric. Eng. Res. 53:23-50 (1992).

As a result of the oligosaccharides present in HM, the SCFA profile of abreast-fed infant is very different from that of a formula-fed infant.For example, breast-fed infants produce virtually no butyrate, withacetate comprising approximately 96% of the total SCFA production.Lifschitz, et al., Characterization of Carbohydrate Fermentation inFeces of Formula-Fed and Breast-Fed Infants, Pediatr. Res. 27:165-169(1990); Siigur, et al., Faecal Short-Chain Fatty Acids in Breast-Fed andBottle-Fed Infants. Acta. Paediatr. 82:536-538 (1993); Edwards, et al.,Faecal Short-Chain Fatty Acids in Breast-Fed and Formula-Fed Babies,Acta. Paediatr. 72:459-462 (1994); Parrett & Edwards, In VitroFermentation of Carbohydrates by Breast Fed and Formula Fed Infants,Arch. Dis. Child 76:249-253 (1997). In contrast, while formula-fedinfants also have acetate (74%) as the major SCFA in feces, they haveconsiderable amounts of propionate (23%) and small amounts of butyrate(3%) present as well. These differences between the SCFA profiles ofbreast-fed infants and formula-fed infants could affect the energy,digestion, and overall health of the formula-fed infant.

Because cow's milk and commercially available infant formulas that arebased on cow's milk provide only trace amounts of oligosaccharides,prebiotics are often used to supplement the diet of formula-fed infants.Prebiotics have been defined as “non-digestible food ingredients thatbeneficially affect the host by selectively stimulating the growthand/or activity of one or a limited number of bacteria in the colon thatcan improve the health of the host”. Gibson, G. R. & Roberfroid, M. B.,Dietary Modulation of the Human Colonic Microbiota-Introducing theConcept of Probiotics, J. Nutr. 125:1401-1412 (1995). Common prebioticsinclude fructo-oligosaccharide, gluco-oligosaccharide,galacto-oligosaccharide, isomalto-oligosaccharide, xylo-oligosaccharideand lactulose.

The incorporation of various prebiotic ingredients into infant formulashas been disclosed. For example, U.S. Patent App. No. 20030072865 toBindels, et al. discloses an infant formula with an improved proteincontent and at least one prebiotic. The prebiotic component can belacto-N-tetaose, lacto-N-fuco-pentaose, lactulose (LOS), lactosucrose,raffinose, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),oligosaccharides derived from soybean polysaccharides, mannose-basedoligosaccharides, arabino-oligosaccharides, xylo-oligosaccharides,isomalto-oligo-saccharides, glucans, sialyl oligosaccharides, andfuco-oligosaccharides.

Similarly, U.S. Patent App. No. 20040191234 to Haschke discloses amethod for enhancing the immune response which comprises administeringat least one prebiotic. The prebiotic can be an oligosaccharide producedfrom glucose, galactose, xylose, maltose, sucrose, lactose, starch,xylan, hemicellulose, inulin, or a mixture thereof. The prebiotic can bepresent in an infant cereal.

Unfortunately, however, there are many disadvantages in theadministration of the above prebiotics to formula-fed infants. Whilethey may beneficially affect the population of probiotics in the gut,they do not produce a SCFA profile that is similar to that of abreast-fed infant. Additionally, the fermentation of many of theseprebiotic substances occurs at a very rapid rate, which often producesexcess gas, abdominal distension, bloating, and diarrhea. Therefore, thechoice of prebiotic substances in infant formulas should be made withthe goal of maximizing potential benefits and minimizing such unwantedside-effects.

Accordingly, it would be beneficial to provide a prebiotic substancethat simulates the functional attributes of human milk oligosaccharidesin infants, such as an increase in the population and species ofbeneficial bacteria in the infant gut and production of a SCFA profilesimilar to that of a breast-fed infant. Additionally, the prebioticsubstance should be well tolerated in infants and should not produce orcause excess gas, abdominal distension, bloating or diarrhea.

SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to a novel methodfor simulating the functional attributes of human milk oligosaccharidesin a formula-fed infant, the method comprising administering atherapeutically effective amount of polydextrose (PDX) to the infant.

The present invention is also directed to a novel method for increasingthe population and species of beneficial bacteria in a formula-fedinfant, the method comprising administering a therapeutically effectiveamount of PDX to the infant.

In another aspect, the present invention is directed to a novel methodfor producing a short-chain fatty acid (SCFA) profile in a formula-fedinfant which is similar to that of a breast-fed infant, the methodcomprising administering a therapeutically effective amount of PDX tothe infant. Specifically, PDX can cause the SCFA profile to have anincreased level of acetate and a decrease in butyrate.

In yet another aspect, the present invention is directed to a novelmethod for decreasing the rate and extent of fermentation of prebioticswithin the gut of a formula-fed infant, the method comprisingadministering a therapeutically effective amount of PDX to the infant.More particularly, the invention reduces the total gas production aswell as the carbon dioxide production within the infant gut.

Among the several advantages found to be achieved by the presentinvention, it is well tolerated in infants and simulates the functionalattributes of human milk oligosaccharides in infants, such as anincreased population and species of beneficial bacteria in the infantgut, optimization of stool characteristics, and production of a SCFAprofile similar to that of a breast-fed infant.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 illustrates total SCFA production during the fermentation of GOS,LOS, PDX2 and FOS.

FIG. 2 illustrates pH changes during the fermentation of GOS, LOS, PDX2and FOS.

FIG. 3 illustrates the relative proportion of acetic acid production inthe fermentation of GOS, LOS, PDX2 and FOS.

FIG. 4 illustrates the relative proportion of propionic acid productionin the fermentation of GOS, LOS, PDX2 and FOS.

FIG. 5 illustrates the relative proportion of butyric acid production inthe fermentation of GOS, LOS, PDX2 and FOS.

FIG. 6 illustrates the relative proportions of acetic acid, propionicacid, butyric acid and total SCFA production in the fermentation of GOS,LOS, PDX2 and FOS.

FIG. 7 illustrates the total SCFA production during the fermentation ofvarious combinations of prebiotic carbohydrates.

FIG. 8 illustrates the pH changes during the fermentation of variouscombinations of prebiotic carbohydrates.

FIG. 9 illustrates the total SCFA production during the fermentation ofdifferent combinations of PDX and GOS.

FIG. 10 illustrates the concentration of acetic acid produced during thefermentation of different combinations of PDX and GOS.

FIG. 11 illustrates the concentrations of propionic acid produced duringthe fermentation of different combinations of PDX and GOS.

FIG. 12 illustrates the concentration of butyric acid produced duringthe fermentation of different combinations of PDX and GOS.

FIG. 13 illustrates the formation of gases as total volume during thefermentation of GOS, LOS, PDX2 and FOS.

FIG. 14 illustrates the formation gases as carbon dioxide concentrationduring the fermentation of GOS, LOS, PDX2 and FOS.

FIG. 15 illustrates the formation of gases as hydrogen concentrationduring the fermentation of GOS, LOS, PDX2 and FOS.

FIG. 16 illustrates the formation of gases as hydrogen disulphideconcentration during the fermentation of GOS, LOS, PDX2 and FOS.

FIG. 17 is a summary of the prebiotic effect of human milk, FOS, LOS,GOS, PDX, and various combinations thereof on fecal microflora.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference now will be made in detail to the embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, not alimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, can be used on another embodiment to yield a stillfurther embodiment.

Thus, it is intended that the present invention covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent invention are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only, and is not intended as limiting the broader aspects ofthe present invention.

Definitions

As used herein, the term “prebiotic” means a non-digestible foodingredient that beneficially affects the host by selectively stimulatingthe growth and/or activity of one or a limited number of bacteria in thecolon that can improve the health of the host.

The term “probiotic” means a microorganism with low or no pathogenicitythat exerts beneficial effects on the health of the host.

As used herein, the term “infant” means a human that is less than aboutone year old.

A “therapeutically effective amount”, as used in the presentapplication, means an amount that provides a prebiotic effect in thesubject.

The term “simulating”, as used herein means having or taking the form orappearance of or having or producing a symptomatic resemblance to.

The terms “functional attributes” mean any inherent quality orcharacteristic that causes something to occur. Examples of functionalattributes of human milk oligosaccharides in the present invention caninclude the increase of the population and species of beneficialbacteria, production of a SCFA profile that is high in acetic acid andlow in butyric acid, and production of a slow rate and low extent offermentation of prebiotics in the gut.

As used herein, the term “infant formula” means a composition thatsatisfies the nutrient requirements of an infant by being a substitutefor human milk. In the United States, the content of an infant formulais dictated by the federal regulations set forth at 21 C.F.R. Sections100, 106, and 107. These regulations define macronutrient, vitamin,mineral, and other ingredient levels in an effort to stimulate thenutritional and other properties of human breast milk.

Invention

In accordance with the present invention, a novel method for simulatingthe functional attributes of human milk oligosaccharides in formula-fedinfants has been discovered. The method involves providing atherapeutically effective amount of PDX to the infant. Theadministration of PDX provides a beneficial effect on the population andspecies of probiotics, produces a SCFA profile that is similar to thatof breast-fed infants and is physically well-tolerated by infants.

PDX is a non-digestible carbohydrate that has been synthesized fromrandomly cross-linked glucose and sorbitol. It is not digested in theupper GI tract and is only partially fermented in the lower GI tract,making it a beneficial ingredient for digestive health. Thephysiological benefits of PDX include increased fecal bulk, reducedtransit time, lower fecal pH and reduced concentration of putrefactivesubstances in the colon. In adults, PDX ingestion has also been shown toaid in the promotion and growth of beneficial bacteria in the intestineand production of SCFAs.

PDX has been identified as a prebiotic substance for adults based on itsfunctions in the GI tract. For example, U.S. Patent App. No. 20040062758to Mayra-Makinen, et al. relates to a composition which comprises aprobiotic and one or more prebiotics, where the prebiotic can be GOS,palatinoseoligosaccharide, soybean oligosaccharide,gentiooligosaccharide, xylooligomers, nondegradable starch,lactosaccharose, LOS, lactitol, maltitol, or PDX. Similarly, U.S. Pat.No. 4,859,488 to Kan relates to a liquid food comprising PDX andoligosaccharides that is useful for curing constipation.

PDX has not, however, been identified as a prebiotic that provides thebenefits of the present invention and can be administered to infants.The gut microflora of infants is well known to be far less developedthan that of an adult. While the microflora of the adult human consistsof more than 1013 microorganisms and nearly 500 species, the gutmicroflora of an infant contains only a fraction of thosemicroorganisms, both in absolute number and in species diversity.Because the bacterial populations and species vary so immensely betweenthe gut of an infant and an adult, it cannot be assumed that a prebioticsubstance that has a beneficial effect on adults would also have abeneficial effect on infants.

In adults, PDX ingestion has been shown to increase the production ofacetate and butyrate. Because butyrate is not noted in appreciablelevels in breast-fed infants and has been associated with harmfuleffects if produced at significant levels in the infant intestine, PDXwould not generally be considered appropriate for infant nutrition basedon its observed effects in the adult GI system. Thus, it was surprisingand unexpected that PDX was actually metabolized primarily to acetateand propionate, with little butyrate formation. Thus, not only did PDXhave a positive impact on the population and species of beneficialbacteria in the infant intestinal tract, but PDX also created a SCFAprofile that was very similar to that of a breast-fed infant and wouldbe extremely well-tolerated by infants.

One particular reference that relates to PDX in the context of infantadministration actually teaches the converse of the present invention.In U.S. Patent App. No. 20030157146 to Rautonen, it is asserted that PDXcan stimulate the immune system of infants. In that application,however, the Applicant discloses that PDX actually decreased thepopulation of Bifidobacteria in the infant gut (Rautonen App., para.0074). Applicant justifies this result by noting that “an abundance ofbifidobacteria may cause also less desirable physiological effects suchas enteric bacterial diseases and immunosuppression.” (Rautonen App.,para. 0069).

Because the reference teaches that PDX actually decreases the populationof Bifidobacteria in the infant gut, it is in direct conflict with theteaching of the present application. Additionally, the reference doesnot demonstrate that PDX increases the production of acetate, decreasesthe production of butyrate or decreases the rate of fermentation ofprebiotics within the infant gut.

In the method of the present invention, a therapeutically effectiveamount of PDX is administered to an infant for the purpose of simulatingthe functional attributes of human milk oligosaccharides. Atherapeutically effective amount of PDX may be between about 1.0 g/L and10.0 g/L, administered daily. In another embodiment, a therapeuticallyeffective amount of PDX may be between 2.0 g/L and 8.0 g/L, administereddaily.

PDX is commercially available from a variety of sources. For example,STA-LITE® PDX is available in 5 lb bags from Honeyville Grain, Inc.,located in Salt lake City, Utah. Alternatively, Litesse® Ultra™ PDX iscommercially available from Danisco Sweeteners, Ltd., located in theUnited Kingdom.

PDX is well-suited for incorporation into an infant formula, as itcontains only 1 Cal/g, as compared to 4 Cal/g for typical prebiotics. Itis also highly soluble and neutral tasting. Therefore, its addition toinfant formula would not change the physical or taste characteristics ofthe composition.

The form of administration of PDX in the method of the invention is notcritical, as long as a therapeutically effective amount is administered.Most conveniently, the PDX is supplemented into infant formula which isthen fed to an infant.

The infant formula for use in the present invention is preferablynutritionally complete and typically contains suitable types and amountsof lipid, carbohydrate, protein, vitamins and minerals. The amount oflipid or fat typically can vary from about 3 to about 7 g/100 kcal. Theamount of protein typically can vary from about 1 to about 5 g/100 kcal.The amount of carbohydrate typically can vary from about 8 to about 12g/100 kcal. Protein sources can be any used in the art, e.g., nonfatmilk, whey protein, casein, casein protein, soy protein, hydrolyzedprotein, amino acids, and the like. Carbohydrate sources can be any usedin the art, e.g., lactose, glucose, corn syrup solids, maltodextrins,sucrose, starch, rice syrup solids, and the like. Lipid sources can beany used in the art, e.g., vegetable oils such as palm oil, soybean oil,palmolein, coconut oil, medium chain triglyceride oil, high oleicsunflower oil, high oleic safflower oil, and the like.

Conveniently, commercially available infant formula can be used. Forexample, Enfalac, Enfamil®, Enfamil® Premature Formula, Enfamil® withIron, Lactofree®, Nutramigen®, Pregestimil®, or ProSobee® (availablefrom Mead Johnson & Company, Evansville, Ind., U.S.A.) may besupplemented with suitable levels of PDX and used in practice of themethod of the invention.

In an embodiment of the present invention, PDX can be administered incombination with another prebiotic. The prebiotic selected can be anyprebiotic known in the art. Examples of prebiotics include, but are notlimited to: FOS, inulin, gluco-oligosaccharide, GOS,isomalto-oligosaccharide, xylo-oligosaccharide, soybeanoligosaccharides, chito-oligosaccharide, gentio-oligosaccharide,manno-oligosacchaide, LOS, lactosucrose, raffinose,aribino-oligosaccharide, glucans, siallyl-oligosaccharide, andfuco-oligosaccharide.

In a particular embodiment of the present invention, PDX is administeredin combination with GOS. GOS is a mixture of oligosaccharides consistingof D-glucose and D-galactose. It is sometimes referred to astrans-galacto-oligosaccharide. It is produced from D-lactose byβ-galactosidase, which can be obtained from Aspergillus oryzae. GOS hasbeen suggested to increase calcium absorption and prevention of boneloss in adults. GOS has been identified as a prebiotic that is usefulfor administration to infants in U.S. Patent App. No. 20030072865 toBindels, et al.

In this embodiment, PDX and GOS can be administered in a ratio ofPDX:GOS of between about 9:1 and 1:9. In another embodiment, the ratioof PDX:GOS can be between about 5:1 and 1:5. In yet another embodiment,the ratio of PDX:GOS can be between about 1:3 and 3:1. In a particularembodiment, the ratio of PDX to GOS can be about 5:5. In anotherparticular embodiment, the ratio of PDX to GOS can be about 8:2.

A therapeutically effective amount of the PDX:GOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, a therapeutically effective amount of the PDX:GOScombination may be between about 2.0 g/L and 8.0 g/L, administereddaily. In a particular embodiment, a therapeutically effective amount ofthe PDX:GOS combination may be about 2 g/L of PDX and 2 g/L of GOS,administered daily.

In another specific embodiment of the present invention, PDX isadministered in combination with LOS. LOS is a semisyntheticdisaccharide formed from D-galactose and D-fructose and joined by aβ-glucosidic linkage. It is resistant to hydrolysis by human digestiveenzymes, but is fermented in the small intestine. It is highly solubleand has a sweet taste. LOS has been identified as a prebiotic that isuseful for administration to infants in U.S. Patent App. No. 20030072865to Bindels, et al. LOS is commercially available from a variety ofsources.

In this embodiment, PDX and LOS can be administered in a ratio ofbetween about 9:1 and 1:9. In another embodiment, the ratio of PDX toLOS can be between about 5:1 and 1:5. In yet another embodiment, theratio of PDX to LOS can be between about 3:1 and 1:3. In a particularembodiment, the ratio of PDX to LOS can about 5:5. In another particularembodiment, the ratio of PDX to LOS can be about 8:2.

A therapeutically effective amount of the PDX:LOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, a therapeutically effective amount of the PDX:LOScombination may be between about 2.0 g/L and 8.0 g/L, administereddaily. In a particular embodiment, a therapeutically effective amount ofthe PDX:LOS combination may be about 2 g/L of PDX and 2 g/L of LOS,administered daily.

In yet another embodiment of the present invention, PDX is administeredin combination with both GOS and LOS. In this embodiment, thePDX:GOS:LOS combination can be administered in a ratio of about50:33:17. Alternatively, the ratio of the PDX:GOS:LOS combination can beabout 1:1:1. In a particular embodiment, the ratio of PDX:GOS:LOS can beabout 1:1.5:1.

A therapeutically effective amount of the PDX:GOS:LOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, a therapeutically effective amount of the PDX:GOS:LOScombination may be between about 2.0 g/L and 8.0 g/L, administereddaily. In an embodiment, a therapeutically effective amount of thePDX:GOS:LOS combination may be about 2 g/L PDX, 2 g/L GOS and 2 g/L LOS,administered daily. In a particular embodiment, a therapeuticallyeffective amount of the PDX:GOS:LOS combination may be about 2 g/L PDX,1.32 g/L GOS and 2.6 g/L LOS, administered daily. In another embodiment,a therapeutically effective amount of the PDX:GOS:LOS combination may beabout 4 g/L PDX, 2.64 g/L GOS and 3.6 g/L LOS, administered daily.

In one embodiment of the invention, PDX can be combined with one or moreprobiotics and administered to an infant. Any probiotic known in the artwill be acceptable in this embodiment. In a particular embodiment, theprobiotic is chosen from the group consisting of Bifidobacterium spp. orLactobacillus spp. In an embodiment, the probiotic is Lactobacillusrhamnosus GG (LGG). In another embodiment, the probiotic isBifidobacterium lactis. In a specific embodiment, the probiotic isBifidobacterium lactis Bb-12, available from Chr. Hansen Biosystems,located in Milwaukee, Wis.

In other embodiments of the present invention, the infant formula maycontain other active agents such as long chain polyunsaturated fattyacids (LCPUFA). Suitable LCPUFAs include, but are not limited to,α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid,eicosapentaenoic acid (EPA), arachidonic (ARA) and docosahexaenoic acid(DHA). In an embodiment, PDX is administered in combination with DHA. Inanother embodiment, PDX is administered in combination with ARA. In yetanother embodiment, PDX is administered in combination with both DHA andARA. Commercially available infant formula that contains DHA, ARA, or acombination thereof may be supplemented with PDX and used in the presentinvention. For example, Enfamil® LIPIL®, which contains effective levelsof DHA and ARA, is commercially available and may be supplemented withLGG and utilized in the present invention.

In one embodiment, both DHA and ARA are administered in combination withPDX. In this embodiment, the weight ratio of ARA:DHA is typically fromabout 1:3 to about 9:1. Alternatively, this ratio can be from about 1:2to about 4:1. In yet another alternative, the ratio can be from about2:3 to about 2:1. In one particular embodiment the ratio is about 2:1.

The effective amount of DHA in an embodiment of the present invention istypically from about 3 mg per kg of body weight per day to about 150 mgper kg of body weight per day. In one embodiment of the invention, theamount is from about 6 mg per kg of body weight per day to about 100 mgper kg of body weight per day. In another embodiment the amount is fromabout 10 mg per kg of body weight per day to about 60 mg per kg of bodyweight per day. In yet another embodiment the amount is from about 15 mgper kg of body weight per day to about 30 mg per kg of body weight perday.

The effective amount of ARA in an embodiment of the present invention istypically from about 5 mg per kg of body weight per day to about 150 mgper kg of body weight per day. In one embodiment of this invention, theamount varies from about 10 mg per kg of body weight per day to about120 mg per kg of body weight per day. In another embodiment, the amountvaries from about 15 mg per kg of body weight per day to about 90 mg perkg of body weight per day. In yet another embodiment, the amount variesfrom about 20 mg per kg of body weight per day to about 60 mg per kg ofbody weight per day.

The amount of DHA in infant formulas for use with the present inventiontypically varies from about 5 mg/100 kcal to about 80 mg/100 kcal. Inone embodiment of the present invention it varies from about 10 mg/100kcal to about 50 mg/100 kcal; and in another embodiment from about 15mg/100 kcal to about 20 mg/100 kcal. In a particular embodiment of thepresent invention, the amount of DHA is about 17 mg/100 kcal.

The amount of ARA in infant formulas for use with the present inventiontypically varies from about 10 mg/100 kcal to about 100 mg/100 kcal. Inone embodiment of the present invention, the amount of ARA varies fromabout 15 mg/100 kcal to about 70 mg/100 kcal. In another embodiment theamount of ARA varies from about 20 mg/100 kcal to about 40 mg/100 kcal.In a particular embodiment of the present invention, the amount of ARAis about 34 mg/100 kcal.

The infant formula supplemented with oils containing DHA and ARA for usewith the present invention can be made using standard techniques knownin the art. For example, they can be added to the formula by replacingan equivalent amount of an oil, such as high oleic sunflower oil,normally present in the formula. As another example, the oils containingDHA and ARA can be added to the formula by replacing an equivalentamount of the rest of the overall fat blend normally present in theformula without DHA and ARA.

The source of DHA and ARA can be any source known in the art. In anembodiment of the present invention, sources of DHA and ARA are singlecell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156; and5,397,591, the disclosures of which are incorporated herein in theirentirety by reference. However, the present invention is not limited toonly such oils. DHA and ARA can be in natural or refined form.

In one embodiment, the source of DHA and ARA is substantially free ofeicosapentaenoic acid (EPA). For example, in one embodiment of thepresent invention the infant formula contains less than about 16 mgEPA/100 kcal; in another embodiment less than about 10 mg EPA/100 kcal;and in yet another embodiment less than about 5 mg EPA/100 kcal. Oneparticular embodiment contains substantially no EPA. Another embodimentis free of EPA in that even trace amounts of EPA are absent from theformula.

The infant formula of the present invention can be prepared using anymethod known in the art. In one embodiment, the PDX is provided inpowder form. It can be mixed with water and other infant formulaingredients in a mixing tank. If GOS and/or LOS are included in theinfant formula, they can be provided in powdered or liquid form. Themixture can then be pasteurized, homogenized and spray-dried to make afinished powder or canned and retorted to make a liquid product.

As an alternative to an infant formula administration, the prebiotic ofthe present invention can be administered as a supplement not integralto the formula feeding. For example, PDX can be ingested in the form ofa pill, tablet, capsule, caplet, powder, liquid or gel. In thisembodiment, the PDX can be ingested in combination with other nutrientsupplements, such as vitamins, or in combination with a LCPUFAsupplement, such as DHA or ARA.

In another embodiment, PDX can be provided in a form suitable forinfants selected from the group consisting of follow-on formula,beverage, milk, yogurt, fruit juice, fruit-based drink, chewable tablet,cookie, cracker, or a combination thereof.

In the method of the present invention, the infant is formula-fed. Inone embodiment the infant is formula-fed from birth. In anotherembodiment, the infant is breast-fed from birth until an age which isless than one year, and is formula-fed thereafter, at which time PDXsupplementation begins.

Human milk oligosaccharides can increase the population and species ofbeneficial bacteria in the intestinal tract, have a SCFA profile that ishigh in acetate and very low in butyrate, and are slowly fermented,avoiding the production of excessive gases. As will be seen in theexamples, the administration of PDX, alone or in combination with otherprebiotics, can be used to increase the population and species ofbeneficial bacteria in the intestinal tract, can preferentially shiftthe SCFA production toward more acetate and propionate production,thereby limiting butyrate production, and can slow down the fermentationrate in the gut so that gas production is limited, minimizing discomfortto the infant. Thus, the administration of PDX, alone or in combinationwith one or more other prebiotics, can simulate the functionalattributes of human milk oligosaccharides in a formula-fed infant.

The following examples describe various embodiments of the presentinvention. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered to be exemplary only, with the scope and spirit of theinvention being indicated by the claims which follow the examples. Inthe examples, all percentages are given on a weight basis unlessotherwise indicated.

EXAMPLE 1

This example illustrates the in vitro fecal fermentation model utilizedin the present invention. The fecal fermentation model in vitro mimicsthe action of the colon microbiota of infants. During fermentation,carbohydrates are consumed and SCFA and gases are produced. Afterfermentation, an analysis of the effect of the prebiotics on thepopulations and species of microorganisms present can be accomplished.

The individual carbohydrates which were studied are set forth in Table1.

TABLE 1 Individual Carbohydrates GOS: Vivinal GOS: Deb. No. 00026961Borculo Domo Ingredients; received Sep. 17, 2002; purity 95.1% LOS:Morinaga Lactulose Anhydride: MLC-A(F), Lot No. FRDL020926; MorinagaMilk Industry Co. Ltd; received Oct. 4, 2002; purity 97% PDX: Sta-LiteIII PDX: Lot No. DZ2K0351913; A. E. Staley FOS: Raftilose P95Fructo-oligosaccharides: Lot No. PCAB022B02; RaffinerieNotre-Dame/Orafti SA; received Sep. 6, 2002; Purity 95.1% PDX2:Litesse ® Ultra ™ PDX: high molecular-weight polymer, max 22 000 MW;Danisco; Lot No. V36020I INU: Raftiline ® HP: long-chain inulin DP ≧ 23(Lot no: hptoh11oh1; Orafti B.V.; received October 2002; D.S. 96.9%,Inulin 99.9%, Sucrose + Fructose + Glucose 0.1%).

Fecal samples were collected from healthy infants aged 2.5-13 months.Five experimental groups were run, using different combinations ofprebiotic carbohydrates in each fermentation group. Twelve babies wererecruited for the Group 1 and 2 fermentations, 17 babies for the Group 3fermentation, 19 babies for the Group 4 fermentation and 23 babies forthe Group 5 fermentation. In groups 1-3, only five babies were able todonate an acceptable sample. The babies recruited for the firstfermentation were 4, 4, 4, 6, 6, 6, 8, 8, 9, 9, 9 and 10 months of age,for the second fermentation 3, 4, 6, 6, 6, 7, 8, 9, 10, 10, 12 and 13months of age, and for the third fermentation 2, 2.5, 3, 4, 4, 4, 4.5,5, 5, 6, 6, 6, 9, 9, 10, 10 and 11 months of age. The ages of the babieswhose samples were used in the fermentation were Group 1: 6, 8, 9, 9, 9months; Group 2: 4, 8, 10, 12, 13 months; and Group 3: 2.5, 5, 6, 10, 11months. In the Group 4 fermentation, 10 babies (of which one baby twice)were able to donate an acceptable sample. The donors for the Group 4fermentation were 2, 2.5, 4, 5, 7, 9, 9, 10, 11 and 15 months of age.For the Group 5 fermentation, twelve babies were able to donate samples,of which the four youngest donors were selected. Thus the donors were 5,6, 6.5 and 6.5 months of age.

Fecal fermentation in vitro was performed according to the method ofKarppinen, which is hereby incorporated by reference in its entirety.Karppinen S., et al., In Vitro Fermentation of Polysaccharides of Rye,Wheat, and Oat Brans and Inulin by Human Faecal Bacteria, J. Sci. FoodAgric. 80:1469-76 (2000).

In the present study, 100 mg of carbohydrate samples were weighed into50 ml bottles and hydrated using 2 ml of carbonate-phosphate buffer atpH 6.9. The samples were kept overnight under anaerobic conditions at 5°C. until preparation of the inoculum. Fecal slurry (12.5 %,weight/volume) was prepared under strictly anaerobic conditions in thesame buffer by pooling fresh infant feces. Eight ml of the suspensionwas dosed to the substrate samples and bottles were closed in theanaerobic chamber giving the final fecal slurry concentration of 10%(weight/volume). Samples were incubated at 37° C. for 1, 2, 4, 8 or 24hours. 0 hour samples were prepared similarly to the centrifugationtubes and frozen rapidly using liquid nitrogen. Fecal blanks withoutadded carbohydrates were included in all fermentation experiments.

Fermentation was finished by removing the bottles from the waterbath andplacing them on ice except prior to gas measurement, when samples werekept at room temperature for immediate sampling. Gas volume was measuredand gas sample (5 ml) was injected to a nitrogenated headspace bottle.The bottle was placed on ice after the sampling. The fermentation samplewas transferred to a centrifugation tube, pH was measured and an aliquot(2 ml) was drawn from the slurry for SCFA analysis and frozen rapidlywith liquid nitrogen.

EXAMPLE 2

This example illustrates the materials and methods necessary todetermine the effectiveness of polydextrose as a prebiotic forformula-fed infants. Specifically, this example illustrates thematerials and methods necessary to analyze SCFAs and gases.

SCFAs were extracted with diethyl ether and analyzed with gaschromatography as described by Karppinen, et al., which is herebyincorporated by reference in its entirety. Karppinen S., et al., InVitro Fermentation of Polysaccharides of Rye, Wheat, and Oat Brans andInulin by Human Faecal Bacteria, J. Sci. Food Agric. 80:1469-76 (2000).Gases (hydrogen, carbon dioxide, methane, hydrogen disulfide, and oxygenas a quality control) were analyzed isothermally at 30° C. using astatic headspace technique by gas chromatography according to Karppinen,et al. Id.

EXAMPLE 3

This example illustrates the effect of PDX on the in vitro SCFA profileproduced by the infant colon microbiota. FIGS. 1 and 2 illustrate thatthe rate of fermentation varies among different prebiotics. Theproduction of total SCFA (a sum of acetic, propionic and butyric acids)is shown in FIG. 1. A decrease in pH, shown in FIG. 2, is also anindication of SCFA production.

As can be seen in the figures, PDX2 is a slowly fermentablecarbohydrate, whereas FOS, GOS and LOS were fermented fast andcompletely. The fermentation rate of PDX2 was comparable to cerealdietary fibers. Not only was PDX2 fermented at the slowest initial rate,but the extent of fermentation was only slightly above the fecal blank.In contrast, the fermentation rate of FOS was so rapid that it wasconsumed almost completely within the first sampling time points andproduced the highest amount of SCFAs among prebiotics tested.

As shown in FIG. 3-5, PDX2 fermentation results in the highestpropionate production and the lowest butyrate production after 24 hours.Acetate was still the highest SCFA produced during the fermentation ofPDX, although the initial rate was much lower than those of the othersubstrates. The initial rate of propionate production from PDX2 wassimilar to that of the other substrates, but higher levels were found atthe end of fermentation. In contrast, the fermentation of FOS, GOS andLOS showed increased concentrations of acetate and butyrate anddecreased concentration of propionate. As a result, the combinedrelative proportion of acetate and propionate was much higher for PDX2than for FOS, LOS or GOS. These results can also be seen in FIG. 6.These results demonstrate that PDX2 was the least butyrate-producingsubstrate and the only substrate for increasing the relative proportionof propionate.

These results are in agreement with an in vitro study conducted by Wang,X. & Gibson, G. R., Effects of the In Vitro Fermentation ofOligofructose and Inulin by Bacteria Growing in the Human LargeIntestine, J. Appl. Bacteriol. 75:373-380 (1993), in which fecal slurryfrom adult donors was used in the fermentation of various carbohydrates.However, the higher propionate production from PDX in vitro was notshown in vivo in a clinical trial with Chinese adults Jie, Z., et al.,Studies on the Effects of Polydextrose Intake on Physiological Functionsin Chinese People, Am. J. Clin. Nutr. 72:1503-09 (2000), in which threedifferent PDX concentrations could increase the levels of butyrate andacetate, but not the proportion of propionate. Larger production ofbutyrate from GOS and FOS has also been shown with human fecal floraassociated rats (Djouzi, Z., et al., Compared Effects of ThreeOligosacchardies on Metabolism of Intestinal Microflora in RatsInoculated with a Human Faecal Flora, Br. J. Nutr. 78:313-24 (1997).

EXAMPLE 4

This example illustrates the effect of combinations of prebiotics on thein vitro fermentation rate by infant colon microbiota. Variouscombinations of prebiotic carbohydrates were chosen in an attempt toachieve a desirable rate of microbial fermentation in vitro. In thisexample, the substrate combinations were compared for their fermentationrate (total SCFA production) and changes in pH, shown in FIGS. 7-8.

The addition of PDX to the GOS preparation slowed the fermentation rateof the combination as measured by total SCFA production (FIG. 7).Similarly, the addition of PDX to the LOS preparation slowed thefermentation rate of the combination. The addition of PDX to LOS or GOSalso resulted in a more moderate decrease in pH, as shown in FIG. 8.This slower rate of acidification of stool content may lead to lessirritation of the intestinal lining or anal region, increasing infanttolerance. The slower decrease in pH by PDX is consistent with slowerSCFA production and overall in vitro fermentation rate compared to GOSand LOS. These results demonstrate that PDX can be used to slow down thefermentation rate of the mixtures of PDX and traditional prebiotics suchas GOS or LOS.

The effect of the PDX:GOS ratio on the production of total SCFA,acetate, propionate and butyrate was also studied (FIGS. 9-12). FIG. 9demonstrates that a PDX:GOS ratio of 8:2 led to a slower rate of totalSCFA production than did a PDX:GOS ratio of 5:5. FIG. 9 confirms that aPDX:GOS ratio of 8:2 produced less total SCFA than a ratio of 5:5 or1:9. Thus, these results demonstrate that a higher amount of PDX in thePDX:GOS mixture results in a slower rate of fermentation in vitro. Theaddition of PDX to GOS also had the tendency to decrease the rate ofacetate and butyrate production, but had little impact on the overallrate and final propionate production.

EXAMPLE 5

This example illustrates the effect of PDX on in vitro gas production byinfant colon microbiota. Total gas production, measured as the totalvolume per fermentation bottle, was about equal with GOS, LOS and FOS,shown in FIG. 13. In contrast, PDX results in lower overall gasproduction during fermentation by infant fecal bacterial microbiota. Thelower overall gas production seen in PDX also indicates that it isfermented more slowly than the other prebiotics studied.

In addition to total gas production, carbon dioxide production is animportant measure of infant tolerance to dietary prebiotics. The majorgas product of all prebiotics tested was carbon dioxide. It was producedin 3- and 44-76-fold higher amounts than hydrogen or hydrogendisulphide, respectively.

Overall, production of carbon dioxide was the lowest for PDX whencompared with FOS, GOS and LOS (FIG. 14). Carbon dioxide was the maingas produced during the fermentation of FOS, GOS and LOS, showingmaximum levels between 320-380 μmol. In contrast, PDX showed much lowerlevels of carbon dioxide formation (200 μmol). Hydrogen formation fromPDX by infant fecal microbiota was lower (about one third) than carbondioxide production, and considerably lower than levels of hydrogenproduced from FOS, GOS and LOS (FIG. 15). Hydrogen disulphide formationfrom PDX was 1:44 compared to the formation of carbon dioxide andmaximal hydrogen disulphide production was at about the same level ofconcentration for all test prebiotics (FIG. 16). The larger proportionof carbon dioxide formation compared to the formation of hydrogen(1000-fold) and methane (10-fold) was also shown by Wang and Gibson.Wang, X. & Gibson, G. R., Effects of the In Vitro Fermentation ofOligofructose and Inulin by Bacteria Growing in the Human LargeIntestine, J. Appl. Bacteriol. 75:373-380 (1993). Since methanogenesiswas not observed in the present study, hydrogen disulphide was formedpresumably from primary hydrogen. Levitt, et al., Gas Metabolism in theLarge Intestine, CRC Press, Boca Raton 131-154 (1995). It is possiblethat hydrogen was not detected due to its further metabolism tosecondary gas, hydrogen disulphide, at late time points.

EXAMPLE 6

This example illustrates the materials and methods necessary todetermine the effect of PDX on the population and species of microbiotafrom the infant colon. Briefly, the example utilizes an infant gut modelto evaluate certain prebiotic compounds. The infant gut in vitro modelutilized, which was based on an adult model, was comprised of two 100 mlglass vessels, arranged in series to represent the proximal and distalregions of the infant colon. The feed flow was controlled at a rate thattook into account the shorter passage time in the infant gut, ascompared to an adult gut. To model in vivo differences in pH within thecolon, vessel 1 (V1) was controlled at pH 5.2 and vessel 2 (V2) wascontrolled at pH 6.7. Temperature was controlled at 37° C. by acirculating water bath. The feed and culture vessels were magneticallystirred and maintained under an anaerobic atmosphere by inflowingoxygen-free nitrogen (15 mL/min).

Once the system was inoculated with infant fecal slurry, the twofermenter vessels were left for up to 24 hours in batch mode. Thisallowed the bacterial populations to equilibrate in their newenvironment and increase in density. The feed flow was then turned onand the fermenter ran in continuous culture mode for the remainder ofthe experiment. The feed flow rate was controlled at 11.11 ml/h. In thisstudy, the fermenters were run for 12 days, 6 days being fed Enfalacinfant formula (Mead Johnson Nutritionals, Evansville, Ind.) and afurther 6 days being fed Enfalac and the added prebiotic or prebioticcombination.

Samples of 5 ml were then taken aseptically from V1 and V2 and preparedfor the culture independent microbial enumeration procedure FluorescenceIn Situ Hybridisation (FISH) and microscopy for the identification andenumeration of specific bacterial species. Using FISH allows theaccurate determination of the effect of prebiotics on specific bacterialpopulations in the proximal and distal regions of the infant colon.

Prebiotics were added to the feed individually or in combinations, at atotal concentration of 7.5 g/l (0.75% w/v). The followingoligosaccharides were used:

TABLE 2 Prebiotics Tested Prebiotic Type Manufacturer Lactulose (LOS)Syrup Morinaga Milk Ind. Co. Ltd., Japan Galacto-oligosaccharide E0002Supplied by Mead (GOS) powder Johnson Polydextrose (PDX) ‘LitesseDanisco Ultra’ powder Fructo-oligosaccharide Raftilose ® Orafti P95powder

The infant donors were carefully selected and ideally aged 2-4 months,formula-fed (exclusively where possible), healthy and not under recentantibiotic treatment. A minimum age of 2 months was preferred as theinfant gut microbiota is established by this age.

TABLE 3 Donor Information Donor Fermentation Code Age Feed Run KB 16weeks SMA Gold F1 JS 13 weeks Cow & Gate F2 F 19 weeks SMA Gold andbreast-fed F3 AE 9½ weeks breast fed F4 AE 14 weeks breast fed F5

The microbial flora of the infant gut for fermentation tests wasprovided by freshly voided infant feces. A fecal sample of at least 3.5g was usually required. The fecal sample was retained in the diaperwhich, immediately on removal from the infant, was placed by thecaregiver into an anaerobic jar with an opened anaerobic gas pack. Thiswas collected and processed as soon as possible (usually within thehour).

In the laboratory, the feces were removed from the diaper and weighed. A10% (w/v) fecal slurry was prepared by homogenizing the samples inanoxic and pre-warmed (overnight in the anaerobic cabinet) 1×PBSsolution, using a stomacher at medium rate for 120 seconds.

Each of the fermenter vessels was inoculated with 5 ml of the 10% w/vfecal suspension. An aliquot of the fecal suspension (sample S) was alsotaken for analysis

A 375 μl sample of the fecal suspension (sample S) or of each fermentersample was required in duplicate for bacterial counts by FISH. Eachsample was fixed by mixing thoroughly in 1.125 ml cold, filtered 4%(w/v) paraformaldehyde solution in PBS pH7.2) and storing at 4° C.overnight (or at least 4 hours).

The fixed sample was centrifuged at 13,000×g for 5 minutes and thesupernatant discarded. The pellet was washed twice by re-suspending in 1ml of cold, filtered 1×PBS, each time pelleting the cells bycentrifugation and discarding the supernatant. The pellet was finallyre-suspended thoroughly in 150 μl of filtered PBS; 150 μl of 96% (v/v)ethanol is then mixed in well. The cell preparation was then stored at−20° C. for at least 1 h before further processing.

In the hybridization step, 16 μl of the cell preparation (brought toambient temperature) was mixed with 200 μl filtered, pre-warmed2×hybridization buffer (30.3 mM Tris-HCl pH 7.2,1.4 mM NaCl) containing15.1 ml/l 10% (w/v) SDS. This mixture was warmed to the appropriatehybridization temperature and then mixed with the probe (50 ng/μl) inthe ratio 9:1, respectively. The hybridization preparation was thenreturned to the hybridization oven to incubate overnight.

Finally, the hybridized cell preparations were collected onto 0.2 μmfilters for microscopic observation. Depending upon the cell density,between 5 μl and 100 μl of the cell preparation was added to filtered,pre-warmed (to hybridization temperature) washing buffer (5-7 ml 20mMTris-HCl pH 7.2, 0.9 M NaCl). 20 μl DAPI (4′,6-diamidino-2-phenylindole)was also added to the mixture to stain all cells and obtain total cellcounts for each sample. This was then vacuum-filtered onto a 0.2 mpolycarbonate filter and placed on a microscope slide. To minimizefading of the fluorescent dye, a drop of SlowFade™ (Molecular Probes)was placed on the filter and covered with a cover slip; the slides werethen stored in the dark at 4° C. until used. Bacteria tagged with a Cy3fluorescent probe were counted using fluorescence microscopy (Leitz,Wetzlar, Germany) at 550 nm; UV light was used for counting DAPI stainedbacteria. Bacteria were counted in at least 15 fields taken at randomand the average of these used to estimate the number of cells per ml ofthe original sample.

Four comparison, fermentation tests were run as listed below.

TABLE 4 Fermentation Runs Fermentation Run Test Substances F1 FOS F2Human Milk PDX F3 GOS F4 1:1 LOS:GOS 1:1 PDX:LOS F5 LOS 1:1 PDX:GOS

EXAMPLE 7

This example illustrates the effect of PDX on the population and speciesof bacteria in the infant gut. In fermentation run 1 (F1), FOS was addedto the formula feed and run in a fermenter system. FOS, which hastraditionally been considered a good prebiotic ingredient, resulted inincreases in Bifidobacteria and Clostridia and decreases in Lactobacilliand Bacteroides in V1. The addition of FOS to formula feed resulted inno change in Bifidobacteria and Lactobacilli levels and increases inClostridia and Bacteroides in V2.

In F2, PDX and human milk were run in parallel fermenter systems. Humanmilk samples were provided by a maternity ward and stored frozen. Thesewere early milk samples of varying volumes from several donors. Thehuman milk feed was run without dilution or addition of lactose in orderto maintain comparable levels of oligosaccharides and other nutrients.There was insufficient human milk to run this fermenter for 12 days, inparallel with the PDX fermenter. More frequent samples were thereforetaken, at days 0, 4, 6 and 8. For comparative purposes, additionalsamples were taken from the PDX fermenter at day 8, and also at day 11.

As would be expected, human milk promoted good growth of beneficialbacteria, both Bifidobacteria and Lactobacilli, and decreased Clostridialevels, as shown in FIG. 17. Bifidobacteria and Lactobacilli clearlyincreased in population in both vessels. Bacteroides numbers remained ata similar level throughout the fermentation.

The results of PDX addition to the formula feed were also favorable,with a marked increase in Lactobacilli and decreases in both Clostridiaand Bacteroides in both vessels (FIG. 17).

In F3, GOS was added to the formula feed and run in a fermenter system.The addition of GOS to the formula feed had little apparent effect onLactobacilli in either vessel, but increased Bifidobacteria in V1 andV2, and decreased Clostridia and Bacteroides in V1 but not V2.

The combination of LOS:GOS (1:1) was run against 1:1 PDX:LOS in aparallel fermenter system during F4. The LOS:GOS combination waseffective in increasing numbers of Lactobacilli in both vessels andBifidobacteria in V1, and in decreasing Bacteroides in V1. Clostridiadecreased in V2 but increased in V1.

Supplementation of the formula feed with a 1:1 combination of PDX:LOSresulted in an increase in Lactobacilli in V1, but a slight decrease inBifidobacteria in each vessel. Clostridia tended to decrease in bothvessels which Bacteroides decreased mainly in V2.

In F5, LOS was supplemented into the formula feed and run in a parallelfermenter system against a 1:1 combination of PDX and GOS. The additionof LOS to the formula feed increased Lactobacilli in both vessels.However, Clostridia also increased in V2 and Bifidobacteria decreased inboth vessels. Although Bacteroides decreased in V1 this was notmaintained in V2. The addition of PDX:GOS to the formula feed increasedlevels of Bifidobacteria and Lactobacilli in both vessels, but alsocaused Clostridia levels to increase. Bacteroides levels increased in V2only.

Overall, Bifidobacteria increased in proportion to the total bacterialpopulation in V1 with human milk, GOS, FOS, PDX and the PDX:GOScombination. In V2, GOS, the PDX:GOS combination and the LOS:GOScombination led to an increase in Bifidobacteria. Clostridia decreasedin proportion to the total population in V1 with human milk, GOS andPDX, and decreased in V2 with human milk, PDX and the LOS:GOScombination.

In V1, the Lactobacilli showed an increase following supplementationwith LOS, PDX, human milk or PDX combinations, whereas increases inLactobacilli were observed in V2 with LOS, PDX, human milk, and GOScombinations. The increases in the percentage of Lactobacilli wereparticularly marked with PDX and the PDX:GOS combination and the LOS:GOScombination in V2.

Overall, PDX was effective in increasing Lactobacilli and decreasinglevels of Clostridia and Bacteroides, with only slight increases inBifidobacteria in V1. The PDX:GOS combination also looked favorable forBifidobacteria, which increased amongst the total bacteria (although notas a percentage of the four groups) and increased Lactobacilli at a pHof 5.2, but it also had the unfavorable effect of increasing Bacteroidesnumbers.

When human milk was tested in the model system designed by theinventors, Bifidobacteria and Lactobacilli levels increased in number,while Clostridia decreased in number. This effect was most consistentlyduplicated with PDX, and with GOS, either alone or in combination withLOS or PDX. FOS, which is another carbohydrate that is currentlyutilized in various infant formulas, was tested and did not produce thesame desirable results.

EXAMPLE 8

This example illustrates one embodiment of an infant formula of thepresent invention.

TABLE 5 Nutrient Information for Infant formula Ingredient Per 10,000 LDemineralized Whey Solids 534.337 kg Fat Blend 339.695 kg Nonfat MilkSolids 191.234 kg Lactose 136.321 kg Galactooligosaccharide Syrup Solid35.096 kg Polydextrose 22.222 kg Potassium Citrate 7.797 kg Mono- andDiglycerides 7.233 kg Single Cell Arachidonic Acid Oil 6.486 kg CalciumPhosphate, Tribasic 4.185 kg Ascorbic Acid 1,403.323 g Sodium Ascorbate1,168.402 g Inositol 407.029 g Taurine 402.962 g Corn Syrup Solids188.300 g Niacimamide 89.857 g Calcium Pantothenate 42.443 g Vitamin B₁₂23.613 g Biotin Trituration 23.613 g Thiamin HCl 8.022 g Pyridoxine HCl6.176 g Folic Acid 2.260 g Lecithin Concentrate 3.694 kg Single CellDocosahexaenoic Acid Oil 3.243 kg Carrageenan 2.826 kg Calcium Chloride2.650 kg Sodium Chloride 1.410 kg Maltodextrin 484.199 g CMP, free acid151.951 g AMP, free acid 33.944 g GMP, disodium salt 18.347 g UMP,disodium salt 7.559 g Ferrous Sulfate 0.620 kg Sodium Citrate 0.455 kgTocopheryl Acetate, DL-Alpha 160.882 g Soy Oil 139.612 g Vitamin APalmitate 17.253 g Cholecalciferol Concentrate 5.715 g Vitamin K, LiquidPhytonadione 0.538 g Zinc Sulfate 214.225 g Sodium Selenite 51.112 gCupric Sulfate 22.885 g Lactose 12.659 g Manganese Sulfate 3.119 gWater, Deflouridated 10,311.900 kg

LOS is generated when lactose is heated at a high temperature.Therefore, in this embodiment the product contains indigenous LOS. Thelevel of indigenous LOS in the product is approximately 2 g/L.

EXAMPLE 9

This example illustrates another embodiment of an infant formula of thepresent invention.

TABLE 6 Nutrient Information for Infant formula Ingredient Per 10,000 LDemineralized Whey Solids 534.337 kg Fat Blend 339.695 kg Nonfat MilkSolids 191.234 kg Lactose 142.000 kg Galactooligosaccharide Syrup Solid23.164 kg Polydextrose 22.222 kg Lactulose Syrup Solid 10.353 kgPotassium Citrate 7.797 kg Mono- and Diglycerides 7.233 kg Single CellArachidonic Acid Oil 6.486 kg Calcium Phosphate, Tribasic 4.185 kgAscorbic Acid 1,403.323 g Sodium Ascorbate 1,168.402 g Inositol 407.029g Taurine 402.962 g Corn Syrup Solids 188.300 g Niacimamide 89.857 gCalcium Pantothenate 42.443 g Vitamin B₁₂ 23.613 g Biotin Trituration23.613 g Thiamin HCl 8.022 g Pyridoxine HCl 6.176 g Folic Acid 2.260 gLecithin Concentrate 3.694 kg Single Cell Docosahexaenoic Acid Oil 3.243kg Carrageenan 2.826 kg Calcium Chloride 2.650 kg Sodium Chloride 1.410kg Maltodextrin 484.199 g CMP, free acid 151.951 g AMP, free acid 33.944g GMP, disodium salt 18.347 g UMP, disodium salt 7.559 g Ferrous Sulfate0.620 kg Sodium Citrate 0.455 kg Tocopheryl Acetate, DL-Alpha 160.882 gSoy Oil 139.612 g Vitamin A Palmitate 17.253 g CholecalciferolConcentrate 5.715 g Vitamin K, Liquid Phytonadione 0.538 g Zinc Sulfate214.225 g Sodium Selenite 51.112 g Cupric Sulfate 22.885 g Lactose12.659 g Manganese Sulfate 3.119 g Water, Deflouridated 10,311.900 kg

LOS is generated when lactose is heated at a high temperature.Therefore, in this embodiment the product contains both added andindigenous LOS. The total level of LOS in the product, including bothadded and indigenous LOS, is approximately 2.6 g/L.

EXAMPLE 10

This example illustrates yet another embodiment of an infant formula ofthe present invention.

TABLE 7 Nutrient Information for Infant formula Ingredient Per 10,000 LDemineralized Whey Solids 534.337 kg Fat Blend 339.695 kg Nonfat MilkSolids 191.234 kg Lactose 119.321 kg Galactooligosaccharide Syrup Solid46.327 kg Polydextrose 44.444 kg Lactulose Syrup Solid 20.706 kgPotassium Citrate 7.797 kg Mono- and Diglycerides 7.233 kg Single CellArachidonic Acid Oil 6.486 kg Calcium Phosphate, Tribasic 4.185 kgAscorbic Acid 1,403.323 g Sodium Ascorbate 1,168.402 g Inositol 407.029g Taurine 402.962 g Corn Syrup Solids 188.300 g Niacimamide 89.857 gCalcium Pantothenate 42.443 g Vitamin B₁₂ 23.613 g Biotin Trituration23.613 g Thiamin HCl 8.022 g Pyridoxine HCl 6.176 g Folic Acid 2.260 gLecithin Concentrate 3.694 kg Single Cell Docosahexaenoic Acid Oil 3.243kg Carrageenan 2.826 kg Calcium Chloride 2.650 kg Sodium Chloride 1.410kg Maltodextrin 484.199 g CMP, free acid 151.951 g AMP, free acid 33.944g GMP, disodium salt 18.347 g UMP, disodium salt 7.559 g Ferrous Sulfate0.620 kg Sodium Citrate 0.455 kg Tocopheryl Acetate, DL-Alpha 160.882 gSoy Oil 139.612 g Vitamin A Palmitate 17.253 g CholecalciferolConcentrate 5.715 g Vitamin K, Liquid Phytonadione 0.538 g Zinc Sulfate214.225 g Sodium Selenite 51.112 g Cupric Sulfate 22.885 g Lactose12.659 g Manganese Sulfate 3.119 g Water, Deflouridated 10,325.600 kg

LOS is generated when lactose is heated at a high temperature.Therefore, in this embodiment the product contains both added andindigenous LOS. The total level of LOS in the product, including bothadded and indigenous LOS, is approximately 3.6 g/L.

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references

These and other modifications and variations to the present inventionmay be practiced by those of ordinary skill in the art, withoutdeparting from the spirit and scope of the present invention, which ismore particularly set forth in the appended claims. In addition, itshould be understood that aspects of the various embodiments may beinterchanged both in whole or in part. Furthermore, those of ordinaryskill in the art will appreciate that the foregoing description is byway of example only, and is not intended to limit the invention sofurther described in such appended claims. Therefore, the spirit andscope of the appended claims should not be limited to the description ofthe preferred versions contained therein.

1-46. (canceled)
 47. A nutritional composition comprising polydextrosein combination with at least one other prebiotic.
 48. The nutritionalcomposition according to claim 55, wherein the amount of polydextrose inthe infant formula is sufficient to deliver between about 1.0 g/L and10.0 g/L of polydextrose to the infant daily.
 49. The nutritionalcomposition according to claim 55, comprising polydextrose andgalacto-oligosaccharide.
 50. The nutritional composition according toclaim 55, comprising polydextrose and lactulose.
 51. The nutritionalcomposition according to claim 55, comprising polydextrose,galacto-oligosaccharide and lactulose.
 52. The nutritional compositionaccording to claim 51, wherein the amount of polydextrose present in theinfant formula is about 2.0 g/L, the amount of galacto-oligosaccharidepresent in the infant formula is about 2.0 g/L and the amount oflactulose present in the infant formula is about 2.0 g/L.
 53. Thenutritional composition according to claim 51, wherein the amount ofpolydextrose present in the infant formula is about 2.0 g/L, the amountof galacto-oligosaccharide present in the infant formula is about 1.32g/L and the amount of lactulose present in the infant formula is about2.6 g/L.
 54. The nutritional composition according to claim 51, whereinthe amount of polydextrose present in the infant formula is about 4.0g/L, the amount of galacto-oligosaccharide present in the infant formulais about 2.64 g/L and the amount of lactulose present in the infantformula is about 3.6 g/L.
 55. The nutritional composition according toclaim 47, which comprises an infant formula.
 56. The nutritionalcomposition according to claim 49, wherein the ratio of polydextrose togalacto-oligosaccharide is between about 9:1 and 1:9.
 57. Thenutritional composition according to claim 47, which further comprisesa. a lipid or fat; b. a protein source selected from the groupconsisting of whey protein, casein, casein protein, nonfat milk,hydrolyzed protein and combinations thereof; and c. about 5 to about 200mg/100 kcal of a source of long chain polyunsaturated fatty acids. 58.The nutritional composition according to claim 57, wherein the source oflong chain polyunsaturated fatty acids comprises docosahexanoic acid,arachidonic acid or combinations thereof.
 59. The nutritionalcomposition according to claim 58, wherein the source of long chainpolyunsaturated fatty acids comprises docosahexanoic acid andarachidonic acid, further wherein the ratio of arachidonic acid todocosahexanoic acid is from about 1:3 to about 9:1.
 60. The nutritionalcomposition according to claim 57, wherein the lipid or fat is presentat a level of about 3 to about 7 g/100 kcal.
 61. The nutritionalcomposition according to claim 57, wherein the protein source is presentat a level of about 1 to about 5 g/100 kcal.
 62. The nutritionalcomposition according to claim 57, which further comprises at least oneprobiotic.
 63. The nutritional composition according to claim 62,wherein the probiotic is selected from the group consisting ofBifidobacteria spp., Lactobacillus spp and combinations thereof.